1. Field of the Invention
The present invention relates to a method for separating and purifying a biomembrane protein from a biomembrane. A biomembrane protein separated and purified at a high purity is important not only for studying the function and structure of a biomembrane protein, but also because it has become useful as a product in the fields of pharmacology, medicine, and engineering. According to the present invention, the desired biomembrane protein can be separated and purified from a biomembrane, with a high purity and without impairing the biological function thereof, by subjecting the biomembrane to gel electrophoresis in the presence of a specified anionic surfactant.
2. Description of the Related Art
A biomembrane is composed mainly of polar lipids and membrane proteins. The membrane proteins maintaining their biological functions are inserted into a bilayer membrane composed of the polar lipid, especially phospholipids.
Most of the biomembrane proteins such as porin of the Escherichia coli outer membrane, cytochrome b.sub.5, (Na.sup.+, K.sup.+)ATPase, (Ca.sup.++)ATPase, and (H.sup.+)ATPase are only slightly soluble in water and, therefore, when these proteins are separated from the biomembrane, the desired biomembrane protein should be solubilized in the first step of the separation and purification operation, unlike water-soluble globular proteins. To solubilize membrane proteins, media having an environment or situation similar to that of the lipid bilayer are required. Various organic solvents and surfactants have been used for the above-mentioned purpose. Typical examples of such organic solvents being acetone, butanol, ethanol, pyridine, and so on and typical examples of such surfactants being anionic surfactants represented by sodium dodecylsulfate, cationic surfactants represented by trimethyldodecyl ammonium chloride, and nonionic surfactants represented by polyoxyethylene dodecyl ether. However, since most organic solvents act as a strong denaturing agent against proteins, it is usually difficult to separate and purify the desired biomembrane protein from the biomembrane without impairing the biological function thereof. Furthermore, since sodium dodecylsulfate, (i.e., "SDS") conventionally used as a typical anionic surfactant in biochemical fields acts as a strong protein denaturing agent, it is usually difficult to separate and purify the desired biomembrane protein without impairing the biological function thereof. Various attempts have been made to solve the above-mentioned difficulties by using, as a medium for solubilizing biomembrane proteins, nonionic surfactants having a low protein denaturation power. However, the critical micelle concentrations of most nonionic surfactants are so low that it becomes difficult to remove the surfactant molecules bound to the protein by dialysis after the separation and purification of the desired biomembrane protein.
Bile acid salts may be used, as an anionic surfactant having a low protein denaturation power, for solubilizing biomembrane protein. However, the bile acid salts or similar natural surface active substances are practically useless in that they are not available in large amounts for commercial or industrial use. On the other hand, cationic surfactants are commonly used as a germicide, since they are strongly bound to lipids constituting the biomembrane when compared with the other surfactants, and since the denaturation power of cationic surfactants against protein is not weak. There are few (or substantially no) cases in which the separation and purification of the biomembrane proteins can be successfully carried out by using cationic surfactants. Thus, these cationic surfactants are not widely used in the separation of the biomembrane proteins.
Various separation and purification methods utilizing the physical or chemical characteristics of proteins have been proposed, such as thermal or pH treating methods, fractional precipitation methods, absorption and desorption methods, chromatographic methods utilizing ion exchanging, isoelectric fractionation methods, density gradient centrifugation methods, electrophoresis methods, affinity chromatographic methods, molecular sieve methods, two phase partition methods, and crystallization methods. These methods have both merits and demerits.